Gene expression profiles can distinguish normal from diseased cells, making expression profiling a standard approach for identifying potential biochemical pathway abnormalities and therapeutic targets. Improvements in the generation of microarrays, newer multiplex probe hybridization techniques, and advances in data analysis have led to increasingly accurate and reproducible results.
However, expression microarrays remain suboptimal in situations where materials used to make hybridization probes are limited and the detection sensitivity is too low. In such circumstances pooled material from several sources or probe amplification techniques have been used, although pooled samples may still cause difficulty in measuring low abundance RNAs without probe amplification. Polymerase chain reaction (PCR)-based amplification methods are highly sensitive but susceptible to amplification artifacts when used to increase very low abundance species, such as tissue-specific transcription factors. A related technique, linear replication, is more specific but much less sensitive. In practice, gene expression analysis with reverse transcription (RT)-PCR is limited to <102 distinct RNA species within a sample. An additional limitation to microarray approaches is that they are prone to false positives when samples contain multiple molecules with similar sequences, such as closely related members of gene families or alternatively spliced gene products.
The frontier of gene expression analysis lies where microarrays and amplification-based detection methods together fall short. This includes samples with high tissue complexity, such as in neurologic, immunologic, or malignant tissues; where pooling of material from multiple samples obscures important biological differences; and where regulatory proteins expressed at low abundance exert large biological effects from small changes in expression level. A single cell contains 105-106 mRNA molecules, while each low abundance species may be present in only a few copies per cell.
There is a need in the art for detection methods that allow for detection of very low abundance mRNA in a sample, without the need for amplification of the mRNA in the sample. The present invention addresses this need.